Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
  • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
  • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
  • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• the invention relates to pleuromutilin derivatives. It concerns the veterinary use of the compound of formula I
• the compound of formula I in free base or in veterinarily acceptable salt form is hereinafter briefly named "the agent of the invention”. It preferably is in salt, especially in hydrochloride salt form. In that form it is known under the generic name valnemulin hydrochloride.
• the compound of formula I in free base form or in chemotherapeutically or veterinarily acceptable salt form is known from e.g. EP 153 277 and its equivalents, e.g. USP 4 675 330, specifically as Example 12 therein.
• Known therefrom is, further,
• an inhibitory activity in vitro against mycoplasms and chlamydia generally, at concentrations of ca. 0.008 to 0.5 ⁇ g/ml;
• mice an inhibitory activity in vivo in mice, using various bacterial strains, and on hens, using mycoplasm strains, at a dosage of ca. 12 to 50 mg/kg body weight;
• the agent of the invention is particularly effective in the therapy of veterinary diseases the expression of which is enhanced by increased stocking density, such as enzootic pneumonia in swine caused by Mycoplasma hyopneumoniae infection, swine dysentery caused by Serpulina (formerly Treponema) hyodysenteriae infection, swine colitis (inflammation of the colon) associated with Serpulina pilosicoli infection, ileitis in swine (porcine proliferative enteropathy; porcine intestinal adenomatosis) associated with Lawsonia intracellularis infection, chronic respiratory disease and arthritis in poultry associated with Mycoplasma gallisepticum infection, secondary pneumonia in swine associated with Pasteurella multocida, Actinobacillus (Haemophilus) pleuropneumoniae and/or Haemophilus parasuis infection, pneumonia in lambs, sheep and cattle (Shipping Fever, Transit Fever, Calf
• the invention thus concerns the veterinary use as defined above. It also concerns the use of the agent of the invention for the manufacture of a medicament for use in the therapy of veterinary diseases the expression of which is enhanced by increasing stocking density.
• a veterinary agent for use in the therapy of veterinary diseases the expression of which is enhanced by increasing stocking density comprising the compound of formula I in free base or in veterinarily acceptable salt form, together with at least one veterinarily acceptable carrier or diluent.
• the animal suffering from veterinary diseases the expression of which is enhanced by increasing stocking density may e.g not already be treated antibacterially with the agent of the invention, or not already be receiving the agent of the invention for growth promotion.
• Mycoplasma hyopneumoniae infection may be diagnosed in conventional manner, e.g as described in veterinary manuals such as Taylor, D.J., in Pig Diseases. 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K. on page 164-165.
• the beneficial activity of the agent of the invention in this use is determined e.g as follows
• Ten field isolates from outbreaks of enzootic pneumonia in different herds and the type strain "J" are included in the test
• the isolates are filter-cloned, identified by the disc growth inhibition test and used in 7th to 10th passage.
• the test for minimal inhibitory concentration (MIC) is performed in liquid medium (Fnis, N.F. et al., Acta Vet. Scand. 32 [1991] 425-429) in tubes of 1 .8 ml containing 2-fold concentration of valnemulin and tiamulin hydrogen fumarate.
• the mycoplasmas are inoculated in 10 fold dilutions, and the cultures read visually for growth using tubes seeded with 10 2 to 10 4 colour forming units.
• MIC is determined as the lowest concentration of test compound showing inhibition of growth compared to control (Friis, N.F. and Szancer, J., Acta Vet. Scand. 35 [1994] 389-394).
• Tissue samples are obtained from the lungs of pigs from various enzootic pneumonia of pigs (EPP)-affected herds. Samples are shipped on dry ice for culture. M. hyopneumoniae is recovered from freshly sectioned lung tissue by direct culture onto Mycoplasma Experience agar. This technique allows the distinctive colonies of M. hyopneumoniae to be recognised and readily separated, by cloning from other faster growing mycoplasma species such as Mycoplasma hyorhinis, often present in EPP lung samples. Isolates are subcultured and identified by disc growth inhibition using specific rabbit antiserum raised to
• M. hyopneumoniae from lung tissue A liquid medium (Mycoplasma Experience Ltd.) containing phenol red and glucose (pH 7.6) is used for the MIC assays.
• test compound Stock solutions of test compound are prepared at 1 mg/ml concentration in deionised water, sterilised by filtration through 0.2 ⁇ pore size membrane filters (Sartorius Minisart, N) and stored at -20°C.
• the stock solutions are diluted in liquid medium to double the final concentrations required.
• MIC tests are carried out according to the method of Tanner & Wu, Avian Diseases 36 (1992) 714-717.
• Actively growing challenge cultures are prepared either from 1 ml aliquots of broth cultures stored at -70°C or from cultures stored on agar at -70°C. The challenge cultures are diluted to give a target titre of 10 3 to 10 s colour changing units/ml.
• 0.1 ml aliquots of challenge inocula are mixed with 0.1 ml aliquots of antibiotic dilution in microtitre wells.
• Each microtitre plate contains uninoculated media at pH 6.8 (M. hyopneumoniae) (end point control) and antibiotic-free inoculated challenge controls. All plates are sealed with adhesive film and incubated aerobically at 36°C. MICs are recorded when the colour change in the challenge control wells match the pH of the end point control. The MIC is the lowest concentration showing no colour change.
• Valnemulin (ch) 0.0005 0.001 0.00025-0.001 0.0025
• Mycoplasma hyopneumoniae reference strain NCTC 10110 obtained from the National Collection of Type Cultures, London, UK
• MEVT G23 a recent field isolate
• the cultures are dispensed in 1 ml aliquots and frozen at -70°C.
• These cultures are used to initiate further broth cultures which are titrated to obtain the number of colour changing units (ecu) in microtitre plates after incubation (36°C).
• Replication allows challenge of antibiotic dilutions with predetermined numbers of ecu in minimal inhibitory concentration (MIC) tests and in the primary passage in habituation studies.
• MIC minimal inhibitory concentration
• a commercially available medium (Mycoplasma Experience Ltd.) containing glucose and phenol red (MEGB) is used at pH 7.6.
• Stock test compound solutions are prepared at 1000 ⁇ g/ml in deionized water. After vortexing, the solutions are sterilized by filtration through 0.2 ⁇ pore size membrane filters (Sartorius Minisart, N). For use in the MIC tests the stock solutions are diluted in mycoplasma broth to double the final concentrations required. In habituation studies the stock solutions are dispensed in 1 ml aliquots and frozen at -20°C. These are thawed and used at 5 day - 7 day intervals to prepare ranges of concentrations of drug (doubling series) in MEGB, in 1.9 ml aliquots, covering the MICs against both strains of M. hyopneumoniae. Oxytetracycline hydrochloride is prepared fresh on each occasion. The dilution ranges are gradually changed from passage to passage to allow for the development of resistance in the mycoplasmas.
• MIC tests are carried out by the method of Tanner & Wu, Avian Diseases 36 (1992) 714-717 before carrying out the habituation study (below) and after the 10th passages 0.1 ml aliquots of the compound dilution are mixed with 0.1 ml aliquots of the challenge inocula, containing between 10 3 and 10 5 colour changing units (ecu) per ml, in microtitre wells.
• Each microtitre plate contains uninoculated medium , medium at pH 6.8 (end-point-control) and drug-free inoculated challenge controls. All plates are sealed and incubated aerobically at 36°C. MICs are recorded when the colour change in the challenge control wells match the pH 6.8 control (orange-yellow). The MIC is the lowest concentration to show no colour change.
• hyopneumoniae given as a homogenate intranasally on three successive days.
• the minimum inhibitory concentration (M IC) of valnemulin against the strain present in material used in Trials 1 & 2 was 0.016 ⁇ g/ml and that for the strain isolated from material used in Trial 3 was 0.0078 ⁇ g/ml.
• the challenge material was originally derived by infection of gnotobiotic pigs with M. hyopneumoniae and subsequent passage in SPF (Specific Pathogen-free) pigs and had been stored below -70°C.
• Trial 1 Medication with valnemulin hydrochloride by stomach tube (gavage) once a day at 0 (control), 2.5, 5, 7.5 or 10 mg/kg body weight per day. Six pigs per group.
• Trial 2 Medication with valnemulin hydrochloride in feed at 0, 100, 200, 300 or 400 ppm (equivalent to 0, 5, 10, 15 and 20 mg/kg/day). Six pigs per group.
• Trial 3 Medication with valnemulin hydrochloride in feed at 0 or 200 ppm (equivalent to 0 and 10 mg/kg/day). Eight pigs per group. Medication was given from the first day of challenge infection until post-mortem examination performed approximately 3 weeks after challenge infection. Disease was assessed by quantifying lung lesions [Cambridge lung lesion scoring system (Goodwin, R.F.W. and Whittlestone, P., J. Hyq. 69 [1971 ] 391 -397), in which the score is an approximation of percent of lung affected with pneumonia], by estimation of lung-bodyweight ratio and by isolation of M.hyopneumoniae from lungs.
• Cambridge lung lesion scoring system Goodwin, R.F.W. and Whittlestone, P., J. Hyq. 69 [1971 ] 391 -397
• Trial 1 Medication by gavage, MIC 0.016 ⁇ g/ml 1>
• Valnemulin thus proves effective for the prevention of experimentally-induced enzootic pneumonia of pigs in separate experiments using challenge material containing two different strains of M. hyopneumoniae.
• the agent of the invention is therefore useful in the therapy of enzootic pneumonia in swine caused by Mycoplasma hyopneumoniae infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• satisfactory results are obtained when the agent is administered at a daily dosage of from about 5 mg/kg to about 15 mg/kg animal body weight, suitably given in divided doses two to four times daily, or in sustained release form.
• the total daily dosage is from about 100 mg to about 1000 mg, preferably from about 100 mg to about 500 mg, given once or twice daily. It may advantageously be administered as sole therapy.
• Preferred doses in drinking water are from 0.01 to 0.05 % weight by volume, particularly 0.01 to 0.025 %, and in feed from 100 to 400 ppm (g/metric tonne), particularly 100 to 200 ppm (g/metric tonne).
• Serpulina hyodysenteriae infection may be diagnosed in conventional manner, e.g. as described in veterinary manuals, such as Taylor, D.J., in Pig Diseases, 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K. on page 143-144.
• the beneficial activity of the agent of the invention in this use is determined e.g. as follows:
• ATCC 31212 Nine field isolates of S. hyodysenteriae from outbreaks of swine dysentery and the type strain, ATCC 31212 are included, and ATCC 29796 (Serpulina innocens, group 3 (Fellstr ⁇ m, C, Res. Vet. Sci. 59 [1995] 1-4). Identification is based on pattern of hemolysis on TSA (Tryticase soy agar) with 5 % bovine blood, and on test for indole production and hippurate hydrolysis (Rosco Diagnostic Tablets, Taastrup, DK).
• the bacteria are transferred from agar plates into 0.9 % saline and turbidity adjusted to 1.0 on the McFarland scale before 10 ⁇ l of each isolate is inoculated on agar plates with two-fold concentrations of valnemulin hydrochloride, tiamulin hydrogen fumarate, dimetridazole, lincomycin hydrochloride, and tylosin. Growth and hemolysis are recorded after 4 days of growth anaerobically and MIC is determined as the lowest concentration of antibiotics where the spirochetes do not grow.
• MIC Minimum inhibitory concentration
• MICs Minimum inhibitory concentrations are determined against 10 strains of S hyodysenteriae isolated from porcine faeces, by the agar dilution method. NCTC (National Collection of Type Cultures) strains or reference strains are used as controls in all MIC tests.
• the agar medium for S. hyodysenteriae consists of BAB2 (Unipath CM271) containing 7 % whole defibnnated sterile sheep blood. All antimicrobial agents are tested in doubling dilutions Prepared antibiotic dilutions are added to an appropriate volume of MH aga r, previously cooled to 50°C , m i xed a nd 20 ml volumes poured . Al l S.
• hyodysenteriae incubations are performed in an anaerobic work station at 37°C (+/- 0.5°C) (Don Whitley Scientific) which provides a strict anaerobic atmosphere comprising 80% nitrogen, 10% hydrogen and 10% carbon dioxide. All other incubations are performed at 37°C. All strains are incubated in an aerobic atmosphere at 37°C for 24 hours The numbers of the organisms are adjusted to an optical density (OD) equivalent to 1 x 10 8 colony forming units (cfu) per ml, using MH broth as a blank.
• OD optical density
• the antibiotic containing plates are inoculated in duplicate using a multipoint inoculator (Denley Instruments) which delivers a plate inoculum of approximately 1 ⁇ l giving an inoculum of 10 4 -10 5 cfu per spot for al l strains (Amon , J. Antimicrob. Chemother. 21 [ 1 988] 701 -71 0; Ericsson et al. , Acta Pathol. Microbiol. et Immunol Scand. 217 [1971] (B) Suppl., 1 -90). Control plates are incubated under the same conditions as the test plates. The MICs are read after 24 and 48 hours, the latter being the definitive reading.
• the MIC is defined as the lowest concentration of antibiotic on which there is no growth, disregarding a single colony or a faint haze caused by the inoculum (National Committee for Clinical Laboratory Standards [1989], Antimicrobial Susceptibility Testing. NCCLC Publications SC3, Villanova, PA, USA)
• the MIC (in ⁇ g/ml) against Serpulina hyodysenteriae were valnemulin (0.1), tiamulin (0.3), lincomycin (50.0), tylosin (200.0) and dimet ⁇ dazole (30.0). All strains of S. hyodysenteriae were susceptible to valnemulin, which was the most active of the agents tested by 3-600 fold. 3. Challenge trial:
• valnemulin hydrochloride fed at 20, 30 and 40 ppm is compared with tiamulin hydrogen fumarate fed at 30 ppm and with unmedicated controls.
• Five groups of 9 (5 to 6 week old) conventionally reared pigs are used for each treatment group.
• the pigs are fed u nm edicated food for 1 4 days , then challenged with the standard strain of S. hyodysenteriae (P18A). Challenge is by the oral route on two consecutive days.
• the medicated feed is introduced on the day after the second challenge.
• a similar procedure is followed except that 4 groups of approximately 9 pigs were used.
• valnemulin hydrochloride fed valnemulin hydrochloride at 5, 10 and 20 ppm and compared to unmedicated controls.
• the pigs used are from an outdoor reared herd and are challenged with a strain of S. hyodysenteriae isolated from an outdoor herd and previously shown to be capable of causing swine dysentery.
• the evidence of clinical disease is assessed daily and a clinical score assigned, rectal swabs are taken twice weekly for isolation of S. hyodysenteriae and all pigs are weighed at regular intervals. Food intake is also recorded.
• a post-mortem examination is carried out 21 days after challenge and the large intestine examined for the presence of lesions. Mucosal scrapings are also taken from 4 areas in the large intestine and cultured for S. hyodysenteriae.
• hyodysenteriae was isolated from rectal swabs taken from all pigs in the control group and also from the 2 affected pigs in the tiamulin treated group. S. hyodysenteriae was also isolated from 2 pigs that received valnemulin fed at 30 ppm, this coincided with the clinical signs of swine dysentery seen subsequent to an incidental infection in this group. S. hyodysenteriae was not isolated from the 20 and 40 ppm groups. There was little difference in the weight gain or feed conversion ratio of all groups fed medicated food.
• valnemulin at inclusion rates down to 10 ppm is effective in preventing the occurrence of clinical signs of swine dysentery, the shredding of spirochaetes in faeces and the presence of lesions at post-mortum examination.
• a few pigs did show clinical signs of swine dysentery but this coincided with an incidental infection which may have affected the intake of antibiotic.
• the effects of concurrent disease on the intake of antibiotic and the incidence of swine dysentery have previously been described (Burch, D.G.S., Vet. Rec. 110 [1982] 244-246). Analysis of weight gain data showed no differences between the treated groups indicating that the antibiotic did not effect palatability of the feed.
• valnemulin did not completely prevent clinical disease or the shedding of spirochaetes but there were significant reductions in clinical scores and the shedding of spirochaetes when compared to the controls.
• tiamulin fed at 30 ppm did not prevent swine dysentery but reduced the incidence of disease when compared to the control group with a reduction in clinical scores.
• in-feed medication with valnemulin at 10 ppm is clearly more effective than Tiamulin at 30 ppm in preventing swine dysentery.
• a group of 60 (3.5 to 4 weeks old) conventionally reared pigs are fed unmedicated food for 14 days then challenged by the oral route with the standard strain of S. hyodysenteriae (P18A).
• P18A standard strain of S. hyodysenteriae
• pigs are allocated to six treatment groups of 8 pigs per group and fed antibiotic containing feed for 10 days, then unmedicated food for a further 14 days.
• Valnemulin hydrochloride is fed at 50, 75, 100 and 150 ppm and compared with tiamulin hydrogen fumarate fed at 100 ppm and with unmedicated controls. Pigs with a range of clinical signs of disease are included in each treatment group.
• the evidence of clinical disease is assessed daily and a clinical score assigned, rectal swabs are taken twice weekly for isolation of
• hyodysenteriae and all pigs are weighed at regular intervals. Food intake is also recorded. A post-mortem examination is carried out 24 days after challenge and the large intestine of each pig examined for the presence of lesions. Mucosal scrapings are also taken from 4 areas in the large intestine and cultured for S. hyodysenteriae.
• control and tiamulin were formed 8 days after challenge.
• the other groups were formed 12, 15, 20 and 28 days after challenge in the following order: valnemulin at 75, 50, 100 and 150 ppm.
• Each group contained pigs that had blood and/or mucus in their faeces as well as animals that only had mild clinical signs for disease, i.e. soft faeces.
• On day 4 of the trial all pigs in the unmedicated control group were showing severe clinical signs of disease and were killed.
• Valnemulin at 50, 75, 100 and 150 ppm thus produced a reduction in the number of days that were needed to recover from clinical swine dysentery when compared to tiamulin. Also animals did not die or require euthanasia in these valnemulin treated groups. There was a significant difference (p ⁇ 0.001 ) in the clinical scores for the valnemulin treated groups compared to the tiamulin group. The shedding of S. hyodysenteriae in faeces was also prevented by all levels of valnemulin after the withdrawal of medicated feed. However, S. hyodysenteriae was isolated from mucosal scrapings from pigs receiving valnemulin at 50 and 75 ppm.
• Tiamulin at 35 and 40 ppm has been shown to eliminate S. hyodysenteriae from faeces but not prevent the recovery from mucosal scrapings (Taylor, D.J., Vet. Rec. 106 [1980] 526-528).
• Tiamulin at 100 ppm has been successfully used under experimental conditions (Taylor, D.J., Proc. 7th IPVS Congress Mexico [1982] 47) to treat swine dysentery in animals that were not so severely affected that they were inappetent.
• tiamulin at 100 ppm reduced mortality compared to unmedicated controls but did not prevent some deaths, this may have been due to the severity of the disease induced by this experimental challenge.
• the agent of the invention is therefore useful in the therapy of swine dysentery caused by Serpulina hyodysenteriae infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• satisfactory results are obtained when the agent is administered at a daily dosage of from about 1 mg/kg to about 5 mg/kg animal body weight, suitably given in divided doses two to four times daily, or ad libitum in feed or water, or in sustained release form.
• the total daily dosage is from about 10 mg to about 400 mg, e.g.
• Preferred doses in drinking water are from 0.001 to 0.05 % weight by volume, particularly 0.001 to 0.005 % , an d i n f e ed f rom 20 to 1 00 g/metric tonne, particularly 20 to 75 g/metric tonne.
• Colitis may be diagnosed in conventional manner, e.g. as described in veterinary manuals such as Taylor, D.J., in Pig Diseases. 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K. on pages 148-149.
• the beneficial activity of the agent of the invention in this use is determined e.g. as follows:
• the results of the MIC determinations for WBHS are shown in Table 6. Generally, the WBHS were susceptible to all of the five antimicrobials. There were no differences in susceptibility between the three groups of WBHS for any of the antimicrobials under test. The MIC values obtained for valnemulin are at the lowest value for 9 out of 10 strains, while they are much higher for the vast majority of the strains with all 4 reference compounds.
• the agent of the invention is therefore useful in the therapy of swine colitis associated with Serpulina pilosicoli infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• the agent is administered at a daily dosage of from about 1 mg/kg to about 5 mg/kg animal body weight, suitably given in divided doses two to four times daily or ad libitum in feed or water, or in sustained release form.
• the total daily dosage is from about 10 mg to about 400 mg, e.g. from about 10 mg to about 200 mg for prevention or about 20 mg to about 400 mg for treatment, given ad libitum in feed or water, or once or twice daily.
• Preferred doses in drinking water are from 0.001 to 0.05 % weight by volume, particularly 0.001 to 0.005 % , and in f eed from 20 to 1 00 g/metric tonne, particularly 20 to 75 g/metric tonne.
• Lawsonia intracellularis infection may be diagnosed in conventional manner, e.g. as described in veterinary manuals such as Taylor, D.J., in Pig Diseases. 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K. on pages 154-157.
• the beneficial activity of the agent of the invention in this use is determined e.g. as follows:
• Monolayers of IEC-18 rat enterocyte cell cultures (ATCC CRL 1589) are established and maintained by standard cell culture methods. One day old monolayers of dividing cells (approx. 30% confluence) are prepared for infection on day 0, on glass coverslips in small culture vials (Tracs).
• Inocula Batches of three strains of Lawsonia intracellularis are used as inocula. These are derived from pigs afflicted with proliferative enteropathy, of the proliferative haemorrhagic enteropathy (PHE) and porcine intestinal adenomatosis (PIA) forms prepared from pig intestines, and passaged in cell cultures as described in the above Lawson reference. The two PHE strains were partly tested for pathogenicity in pigs as described in McOrist, S. et al., Infect. Immun. 61 (1993) 4286-4292. Inocula batches are collected after several cell passages, and stored frozen at -70°C in 1 ml vials. Preliminary trials established appropriate dilutions of thawed vials that would result in readily recognizeable cell infections in 5 day trials.
• PHE proliferative haemorrhagic enteropathy
• PIA porcine intestinal adenomatosis
• Antibiotics are prepared as 100x stock solutions for each use. Activity of each batch of antibiotic is confirmed by standard Kirby-Bauer disc methods with a laboratory strain of Escherichia coli.
• this inoculum is initially incubated for one hour at 37°C in culture medium containing the antibiotic at various concentrations prior to addition to cells.
• this inoculum is prepared in culture medium, containing the antibiotic at various concentrations added immediately prior to time of addition to cells.
• HIC in control (group 1 ), intracellularly active antibiotic assay (group 2), extracellularly active antibiotic assay (group 3) and continuous treatment assay (group 4) are compared in triplicate assays, for up to 3 strains each. Some assays are repeated for various inocula strengths. Percentage ratios are calculated after derivation of the mean HIC of the controls relevant to the test group. Mean control HIC is the total number of HIC in control Tracs divided by the number of infected control Tracs. The percentage ratio of test Tracs is then calculated by dividing their HIC value by the mean control HIC, multiplied by 100 to give a percentage. That is, test Tracs where antibiotics has no effect would have a percentage ratio of around 100 and where antibiotics completely inhibits growth, the ratio would 0.
• the agent of the invention is therefore useful in the therapy of ileitis in swine associated with Lawsonia intracellularis infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• the agent is administered at a daily dosage of from about 1.5 mg/kg to about 6.5 mg/kg animal body weight, suitably given ad libitum in water or feed, or in divided doses two to four times daily, or in sustained release form.
• the total daily dosage is from about 10 mg to about 1000 mg, preferably from about 15 mg to about 500 mg, given once or twice daily.
• Preferred doses in drinking water are from 0.001 to 0.05 % weight by volume, particularly 0.001 to 0.005 % , and i n feed f rom 20 to 400 g/metric tonne, particularly 20 to 200 g/metric tonne.
• Infection may be diagnosed in conventional manner, e.g., for Mycoplasma gallisepticum infection, as described in veterinary manuals such as Diseases of Poultry, 8th Ed. (1984), Ed. Hofstad et al., Iowa State University Press, on pages 196-198.
• the beneficial activity of the agent of the invention in this use is determined e.g. as follows:
• valnemulin hydrochloride The antimicrobial effect of valnemulin hydrochloride is determined by means of a standard serial microdilution technique in comparison to tylosin (Tylan soluble) and tiamulin hydrogen fumarate.
• test compound 12.8 mg are dissolved in 100 ml of distilled water to obtain a stock solution which is stored at -20°C until used.
• Mycoplasma gallisepticum reference strain used are X95, S6 Holland, S6 Bench (England), MS-16 (Japan), MK-7 (Japan) and 1226, and various fresh field isolates.
• the antibiotic-sensitivity of the strains is examined using a modified microbroth dilution procedure (Stipkovits et al., Vet. Microbiol. 15 [1987] 65-70). Testing medium is added (100 ⁇ l) to all the wells of microtitration plates, using doubling dilutions of antibiotics.
• the inoculum (100 ⁇ l) contains 10 5 cfu/ml.
• the plates are sealed with sellotape and incubated at 37°C over a period of 3 days.
• the lowest concentration of the antibiotic completely preventing colour change of the media at the third day reading is the minimal inhibitory concentration (MIC).
• the wells without colour range are checked for viability of mycoplasmas by plating them on agar medium.
• All Mycoplasma strains are propagated in 10 ml of medium B (Ern ⁇ , H. and Stipkovits, L, Acta Vet. Scand. 14 [1973] 436-449). After incubation at 37°C until early log phase of growth, 1 ml aliquots of culture are dispensed in tubes as seed cultures and stored at -20°C.
• the glucose fermenting Mycoplasma strains are tested in modified medium Bg supplemented with 1 % glucose (pH 7.8), the arginine hydrolysing strains in medium Ba (Ern ⁇ , H. and Stipkovits, L., supra. 450-463) containing 1 % arginine (pH 7.3) and the glucose- and arginine-negative strains in medium B including 1% triphenyltetrazolium chloride.
• iesults obtained with reference mycoplasma strains indicate that the three show more or less the same activity range.
• MIC values of avian field isolates reflect a pronounced difference between the test substances: whereas a uniformly high sensitivity against valnemulin (0.03-0.06 ⁇ g/ml) is displayed, the sensitivity against tiamulin is somewhat reduced (0.06-1 ⁇ g/ml), and with tylosin even resistant strains are obvious (0.06-32 ⁇ g/ml).
• Groups of male broiler chickens are infected with a virulent strain of Mycoplasma gallisepticum (MG) and each group medicated respectively with one of the following: valnemulin (concentrations of 0.00312, 0.00625, 0.0125 and 0.025%), tiamulin (0.00625, 0.0125 and 0.025%), and tylosin (0.05%).
• Infection is effected on day 2 by injection of 0.1 ml of Mycoplasma gallisepticum culture containing approximately 10 6 cfu of viable organisms into each lung (Jordan, F.T.W., The Veterinary Record 127 [1990] 502) and medication is commenced within an hour of infection and continued for three consecutive days. There is also an infected untreated group and an uninfected group.
• Groups of male broiler chicks are infected with a virulent strain of Mycoplasma gallisepticum (MG) and each group medicated respectively with valnemulin at a concentration of 0.0125%, 0.025%, 0.05%, tiamulin at the same concentration, tylosin 0.05% and a lincospectin, spectinomycin combination (Linco-Spectin) 0.083%.
• MG Mycoplasma gallisepticum
• valnemulin at a concentration of 0.0125%, 0.025%, 0.05%, tiamulin at the same concentration, tylosin 0.05% and a lincospectin, spectinomycin combination (Linco-Spectin) 0.083%.
• the chicks are infected at two days of age by the injection of 3.4 x 10 5 organisms into each lung. Medication in the drinking water is
• Results obtained show that control of mortality, with no more than 5%, was best with all three concentrations of valnemulin, and with tiamulin at 0.05%. Lesions were not seen in chicks medicated with the highest and lowest doses of valnemulin and in only a few receiving tiamulin and tylosin. Weight gains for valnemulin at all concentrations were higher than for the tiamulin treated groups and equal to the tylosin group. Mycoplasma were not recovered, at the termination of the experiment at 23 days, from the 3 groups of birds on valnemulin and those given tylosin. In the serological results, at this time, there were some positive reactors in all infected groups.
• the agent of the invention is therefore useful in the therapy of chronic respiratory disease and arthritis in poultry associated with Mycoplasma gallisepticum infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• satisfactory results are obtained when the agent is administered in drinking water at dosages of from about 0.005 to about 0.05 % weight by volume, particularly 0.01 to 0.03 %, and in feed from 20 to 400 g/metric tonne, particularly 20 to 200 g/metric tonne.
• the minimal inhibitory concentration (MIC) of valnemulin hydrochloride against recent isolates of Pasteurella multocida, Haemophilus pleuropneumoniae and Haemophilus parasuis originating from porcine respiratory material is determined and compared with tiamulin.
• Cultures of bacterial isolates comprising 10 P. multocida, 10 H. pleuropneumoniae and 3 H. parasuis are used, isolated within the last 5 years from diseased pigs as assessed at post-mortem examinations.
• Three of the Pasteurella isolates were toxigenic strains of Pasteurella multocida. For H. pleuropneumoniae and H.
• 18 ml of molten agar (50°C) is mixed with 2 ml (one in ten dilutions) of each of the prepared dilutions of test compound in 90 mm Petri dishes.
• Four replicate plates are prepared from each dilution.
• 4 replicate plates containing 18 ml of agar and 2 ml of sterile distilled water are also prepared to act as growth indicators.
• the dried MIC plates are inoculated with 0.2 ⁇ l of the prepared test organisms, those with P. multocida are incubated in air at 37°C for 1 8 hours, and those inoculated with H. pleuropneumoniae and H. parasuis are incubated at 37°C for 18 hours in 10 % CO 2 .
• After inoculation the growth of the organisms is examined initially by eye followed by microscopic examination on a stereo zoom microscope. The MIC value is taken as the lowest concentration of test compound at which no growth can be detected at the point of inoculation on all four plates by microscopic examination. The results obtained are as appears from the Table 10:
• the agent of the invention is therefore useful in the therapy of secondary pneumonia in swin e associated with Pasteurella m ultocida, Actinobacillus ( Haemophi lus) pleuropneumoniae and/or Haemophilus parasuis infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time
• the agent is administered at a daily dosage of from about 5 mg/kg to about 15 mg/kg animal body weight, suitably given ad libitum in water or feed, or in divided doses two to four times daily, or in sustained release form
• the total daily dosage is from about 100 mg to about 1000 mg, preferably from about 100 mg to about 500 mg, given once or twice daily.
• Preferred doses in drinking water are from 0.01 to 0.05 % weight by volume, particularly 0 01 to 0.025 %, and in feed from 100 to 400 ppm (g/met ⁇ c tonne), particularly 100 to 200 ppm
• Pasteurella haemolytica infection may be diagnosed in conventional manner, e.g. as described in veterinary manuals such as Veterinary Medicine. 8th Ed. (1 994) , Eds. Radostits, O.M., Blood, D.C. and Gay, C.C., Publ. Bailhere Tmdall, London, U.K , on pages 748-770.
• the beneficial activity of the agent of the invention in this use is determined in vivo e.g as follows
• the clinical efficacy is determined of two formulations of valnemulin hydrochloride in the treatment of pneumonia in calves due to a controlled infection with Pasteurella haemolytica A1
• 24 calves that have been nasal swabbed and found to be free of P. haemolytica A1 are weighed and allocated to four treatment groups of six, balanced according to live weight with an even distribution of sexes between the groups
• the calves are housed in individual pens in a conventional calf rearing unit where they acclimatise for 33 days before allocation. Blood samples are taken twice during the pre-study phase for leukotoxin antibody analysis as a possible indication of prior exposure to P. haemolytica A1
• Treatment commences on day -1 , approximately 27 hours before the time of challenge Group 1 - Control;
• Group 2 - valnemulin hydrochloride 2.5 % injectable (2.5 mg/kg twice daily)
• Injection sites are also examined Any calves that are recumbent, and/or showing depression and/or signs of respiratory distress, or which have a total clinical score greater than 5 at a single examination between days 0 - 3 are euthanased immediately on humane grounds by intravenous administration of a lethal dose of Pentobarbitone Sodium BP(Vet) 20 % w/v and necropsied within 24 hours. Calves that survive to day 4 after P. haemolytica challenge are euthanased on day 4 following the same procedure as for those euthanased between days 0 - 3.
• Lungs are removed from all calves, and assessed visually for consolidated lesions and pleuritic adhesions
• Lung tissue samples are excised from eight standard sites and tested for the presence of P haemolytica to determine a value for isolation index.
• Heart blood swabs are taken from calves that die acutely and tested for the presence of P haemolytica.
• the agent of the invention is therefore useful in the therapy of pneumonia in lambs, sheep and cattle associated with Pasteurella haemolytica infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• satisfactory results are obtained when the agent is administered at daily dosages of from about 5 mg/kg to about 15 mg/kg animal body weight, suitably given ad libitum in feed, or in divided doses two to four times daily, or in sustained release form.
• the total daily dosage is from about 250 mg to about 3000 mg, preferably from about 250 mg to about 1000 mg, given once or twice daily, or administered ad libitum in feed.
• Mycoplasma hyosynoviae infection may be diagnosed in conventional manner, e.g. as described in veterinary manuals such as Taylor, D.J., in Pig Diseases. 6th Ed. (1995), Publ. D.J. Taylor, Glasgow, U.K., pages 172-173.
• the beneficial activity of the agent of the invention in this use is determined e.g. as follows:
• Tissue samples are obtained from the lungs of pigs from various enzootic pneumonia of pigs (EPP) - affected herds. Samples are shipped on dry ice for culture. Four isolates from lung samples initially isolated for recovery of Mycoplasma hyopneumoniae were reisolated for Mycoplasma hyosynoviae after it had been noted from the lung samples submitted (restored at -70°C), during culture for M. hyopneumoniae, that some samples yielded luxuriant growth of colonies having the typical morphology of Mycoplasma hyosynoviae. Three further isolates were recovered from other lung samples provided.
• test compound Stock solutions of test compound are prepared at 1 mg/ml concentration in deionised water, sterilised by filtration through 0.2 ⁇ pore size membrane filters (Sartorius Minisart, N) and stored at -20°C.
• the stock solutions are diluted in liquid medium to double the final concentrations required.
• MIC tests are carried out according to the method of Tanner and Wu, Avian Diseases 36 (1992) 714-717.
• Actively growing challenge cultures are prepared either from 1 ml aliquots of broth cultures stored at -70°C or from cultures stored on agar at -70°C. The challenge cultures are diluted to give a target titre of 10 3 to 10 5 colour changing units/ml.
• 0.1 ml aliquots of challenge inocula are mixed with 0.1 ml aliquots of antibiotic dilution in microtitre wells.
• Each microtitre plate contains uninoculated media at pH 7.6 (end point control) and antibiotic-free inoculated challenge controls. All plates are sealed with adhesive film and incubated aerobically at 36°C. MICs are recorded when the colour change in the challenge control wells matches the pH of the end point control. The MIC is the lowest concentration showing no colour change.
• the agent of the invention is therefore useful in the therapy of polyarthritis in swine associated with Mycoplasma hyosynoviae infection.
• the effective dosage will, of course, vary depending on the particular salt employed, the mode of administration, the size and age of the animal and the effect desired; for example for prophylactic treatment relatively low doses would be administered over a long time.
• satisfactory results are obtained when the agent is administered at a daily dosage of from about 5 mg/kg to about 15 mg/kg animal body weight, suitably given ad libitum in water or feed, or in divided doses two to four times daily, or in sustained release form.
• the total daily dosage is from about 100 mg to about 1000 mg, preferably from about 100 mg to about 500 mg, given once or twice daily.
• Preferred doses in drinking water are from 0.01 to 0.05 % weight by volume, particularly 0.01 to 0.025 %, and in feed from 100 to 400 ppm (g/metric tonne), particularly 100 to 200 ppm (g/metric tonne).
• the compound may be used in free base form or in veterinarily acceptable salt form, e.g. quaternary salt or, especially, acid addition salt form.
• Such salt forms exhibit the same order of activity as the free base form.
• suitable acid addition salts are the hydrogen fumarate, fumarate, naphthalin-1 ,5-sulphonate and especially the hydrochloride.
• the agent of the invention may be administered orally, locally or parenterally and admixed with conventional chemotherapeutically acceptable diluents and carriers and, optionally, other excipients and administered in such forms as tablets, capsules or injectable preparations. It also forms an excellent additive for feed mixes (as premix) or for drinking water.
• Preferred veterinarily acceptable carriers include e.g. commonly used pharmaceutical excipients like sugar, corn starch, lactose, cellulose, as well as grain carrier systems and grain by-products like ground rice hulls, wheat middlings and soy flour; furthermore, solid diluents like ground limestone, sodium sulfate, calcium carbonate and kaolin, or liquid substances like veterinarily acceptable oils (vegetable oils or mineral oil), propyleneglycol and polyetheleneglycol.
• these formulations are administered to the animal orally, preferably mixed into feed in form of medicated meal feed or medicated pellets.
• solutions in water with or without solvents such as ethanol, propyleneglycol, polyethyleneglycol, approved liquid surfactants and sorbitol are used.
• the formulation is typically prepared as a solution in water which may contain solvents like ethanol or propyleneglycol, or in a veterinarily acceptable oil.
• acceptable oils are sesame oil, medium chain triglycerides (e.g. Miglyol), isopropyl myristate and ethyl oleate.
• the formulation may contain preservatives, buffers and other common excipients.
• Veterinary formulations for use in the present invention may be prepared by mixing the ingredients in the required proportions. The formulation is then packaged into an appropriate container ready for administration.
• the following Example illustrates the invention:
• the agent of the invention is well tolerated.
• the acute toxicity in the rat of the compound of formula I in hydrochloride salt form was determined with single doses of 1000 mg/kg and 2000 mg/kg orally administered to 5 male and 5 female rats per dose group. Deaths occurred within 1 -8 days.
• the LD 50 -value obtained is > 1000 mg/kg p.o.

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